Three-demensional ex vivo angiogenesis system

ABSTRACT

An in vitro tissue angiogenesis and vasculogenesis system is disclosed that allows the outgrowth of microvessels from a three-dimensional tissue fragment implanted in a matrix. The matrix may, for example, be a fibrin- or collagen-based matrix fed by a growth medium, for example, a mixture of tissue culture medium, serum, or a layer of growth medium containing a defined mixture of growth factors. This system, which may be used with human or other mammalian or animal tissues, may be used in assaying tumor angiogenic potential, or in promoting angiogenesis in other tissues, e.g., promoting angiogenesis prior to transplantation of a tissue. The angiogenic potential of a tissue can be determined by measuring the growth of microvessels into the matrix. The three-dimensional structure of the tumor or other tissue is maintained in the matrix, including blood vessels. In another aspect, the method allows for the proliferation of a tissue specimen, thus increasing the mass of cells available for subsequent transplant; and the method also provides for the proliferation of blood vessels from the tissue mass, thus enhancing the chance of successful engraftment.

[0001] The benefit of the May 30, 2000 filing date of provisionalapplication α______ (which is a conversion of nonprovisional application09/580,894) is claimed under 35 U.S.C. § 119(e).

[0002] The development of this invention was subject to a contractbetween the Board of Supervisors of Louisiana State University andAgricultural and Mechanical College, and the United States Department ofVeterans Affairs. The Government has certain rights in this invention.

[0003] This invention pertains to methods to promote ex vivoangiogenesis in tissues, for example, in tissues to be transplanted.This invention also pertains to methods to assay angiogenesis intissues, for example tumor tissues, and to assess the effects ofinducers and inhibitors of angiogenesis. Such information can be of use,for example, in making a prognosis for a tumor, or in evaluating thelikely effect in vivo of anti-angiogenic factors on a tumor.

[0004] “Neovascularization,” “vasculogenesis,” and “angiogenesis” areterms that describe the formation of new capillaries. Angiogenesis is anormal physiological process, the generation of new capillary bloodvessels from pre-existing vessels. Angiogenesis rarely occurs inphysiologically normal adult tissues. Exceptions include the ovary, theendometrium, the placenta, wound healing, and inflammation. Angiogenesisis an important step in ovulation and also in implantation of theblastula after fertilization. Angiogenesis is sometimes distinguishedfrom vasculogenesis, the emergence of blood vessels de novo from asubpopulation of mesenchymal cells known as angioblasts, whichdifferentiate into endothelial cells.

[0005] The identification of several angiogenic factors and theisolation and culture of capillary endothelial cells (ECs) have led to agreater understanding of the cellular and biochemical bases of newvessel growth. Until recently ECs have been the focus of most studies ofmicrovascular growth. However, capillaries are not simply tubes of ECs;they also contain a second cellular component, the mural cell, orpericyte. Angiogenesis involves the differential growth and sprouting ofendothelial tubes, and the recruitment and differentiation ofmesenchymal cells into vesicular smooth muscle cells and pericytes.Communication between the endothelium and the mesenchyme is importantfor angiogenesis. Three such communication pathways have beenidentified:

[0006] (1) Mesenchymal cells signal endothelial cells via theangiopoietin/Tie-2 signaling pathway. See Suri et al., Cell 87: 1171(1996); T. Sato et al. Nature 376, 7074 (1995); Maisonpierre et al.,Science 277: 55 (1997).

[0007] (2) Endothelial cells induce differentiation of pericytes throughthe platelet-derived growth factor (PDGF) signaling pathway. See Lindahlet al. Science 277: 242 (1997); Soriano, Genes Dev. 8: 1888 (1994).

[0008] (3) An endoglin-mediated pathway of endothelial-mesenchymalcommunication was reported by Li et al. Science. 284: 1534-1537 (1999).

[0009] In normal adult mammals, angiogenesis occurs infrequently, yet itcan be rapidly induced in response to various stimuli. The normal rateof capillary endothelial cell turnover in adult mammals is typicallymeasured in months or years. However, when the normally quiescentendothelial cells lining venules are stimulated, they will degrade theirbasement membrane and proximal extracellular matrix, migratedirectionally, divide, and organize into new functioning capillarieswith new basal lamina within a matter of days. This dramaticamplification of the microvasculature of a tissue is temporary, for asrapidly as they are formed the new capillaries virtually disappear,returning the tissue's vasculature to its previous state.

[0010] Among the most extensively studied of normal angiogenic processesis wound repair. Important characteristics of wound-associatedangiogenesis are that it is local, rapid, transient, tightly controlled,and that it promptly regresses back to a steady-state level. The abrupttermination of angiogenesis following wound repair apparently resultsfrom two control mechanisms, mechanisms that are not mutually exclusive.First, due to factors that are not well understood, there appears to bea marked reduction in the synthesis or elaboration of angiogenicmediators. Second, there appears to be a simultaneous increase in levelsof substances that inhibit new vessel growth. The control ofangiogenesis thus depends on a balance of several positive and negativeregulators.

[0011] Recent research has begun to uncover the genetic mechanismscontrolling angiogenesis. See Maswell et al. Nature 399, 271-275 (1999);Stebbins et al., Science 284, 455-461 (1999); Kaumra et al. Science 284,662-665.

[0012] Angiogenesis is regulated by both angiogenic and angiostaticfactors. The role of inhibitors in angiogenesis was first suggested byobservations that hyaline cartilage appeared to be particularlyresistant to vascular invasion. It was later observed that many othercell and tissue extracts also contain inhibitors of angiogenesis.Several natural and artificial angiogenic inhibitors have beenidentified, including: inhibitors of basement membrane biosynthesis,placental RNase inhibitor, lymphotoxin, interferons, prostaglandinsynthetase inhibitors, heparinbinding fragments of fibronectin,protamine, angiostatic steroids, several anti-neoplastic andanti-inflammatory agents, platelet factor-4, thrombospondin-1,angiostatin, integrin antagonists, and certain forms of thrombin.

[0013] Gasparini, Drugs July;58(1):17-38(1999) discusses the possibleuse of angiogenesis inhibitors to intervene into neoplastic processes.The basic idea is to use inhibitory agents to block angiogenesis,thereby causing tumor regression in various types of neoplasia.Therapeutic candidates include naturally occurring angiogenesisinhibitors (e.g., angiostatin, endostatin, platelet factor-4), specificinhibitors of endothelial cell growth (e.g., TNP-470, thalidomide,interleukin-12), agents that neutralize angiogenic peptides (e.g.,antibodies to fibroblast growth factor or vascular endothelial growthfactor, suramin and its analogs, tecogalan, agents that neutralizereceptors for angiogenic factors, agents that interfere with vascularbasement membrane and extracellular matrix (e.g., metalloproteaseinhibitors, angiostatic steroids), and anti-adhesion molecules (e.g.,antibodies such as anti-integrin alpha v beta 3). Rosen L, Oncologist; 5Suppl 1:20-7 (2000) discusses strategies for the application ofantiangiogenic therapies to cancer.

[0014] Other compounds that have been described as inhibitors ofangiogenesis include the cartilage-derived inhibitor TIMP,thrombospondin, laminin peptides, heparin/cortisone, minocycline,fumagillin, difluoromethyl omithine, and sulfated chitin derivatives.

[0015] Of particular interest is the new class of antiangiogenicsubstances called METH proteins. Their enzymatic activity makes thisclass of agents candidates for possible control by small molecules, agoal that has eluded pharmacotherapy. See Vazquez F. et al. J Biol ChemAug 13;274(33):23349-57 (1999). The angiotensin II type 2 receptor isanother example of a receptor that mediates an antiangiogenic response,and that may be amenable to regulation by small molecules.

[0016] Hypoxic conditions can induce angiogenesis. Conversely, whennewly-formed vessels bring oxygen to the tissue, the proteins involvedin induction of angiogenesis are marked for destruction and angiogenesisceases.

[0017] Numerous factors have also been identified that induce vesselformation in vitro or in vivo in animal models. These include: αFGF,βFGF, TGF-α, TNF-α, VPF, VEGF, PDGF, monobutyrin, angiotropin,angiogenin, hyaluronic acid degradation products, and AGE-products.

[0018] Monitoring angiogenic processes can provide valuable informationon tumor progression, metastasis and prognosis (Szabo and Sandor, Eur JSurg Suppl;(582):99-103 (1998)). There is an unfilled need for improvedmethods of monitoring angiogenesis to support the development andapplication of antiangiogenic interventions. The ability to monitorangiogenesis will also assist the discovery of new antiangiogenicagents.

[0019] Diseases Associated with Angiogenesis.

[0020] Abnormal angiogenesis occurs when improper control ofangiogenesis causes either excessive or insufficient blood vesselgrowth. For example, conditions such as ulcers, strokes, and heartattacks may result in some cases from levels of angiogenesisinsufficient for normal healing. Conversely, excessive blood vesselproliferation may favor tumor growth and spread, blindness, andarthritis. Diseases that have been associated with neovascularizationinclude, for example, diabetic retinopathy, macular degeneration, sicklecell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagetsdisease, vein occlusion, artery occlusion, carotid obstructive disease,chronic uveitis/vitritis, mycobacterial infections, Lyme disease,systemic lupus erythematosis, retinopathy of prematurity, Eales disease,Bechets disease, infections causing retinitis or choroiditis, presumedocular histoplasmosis, Bests disease, myopia, optic pits, Stargartsdisease, pars planitis, chronic retinal detachment, hyperviscositysyndrome, toxoplasmosis, trauma, and post-laser complications. Otherangiogenic-related diseases may include, for example, diseasesassociated with rubeosis (neovascularization of the angle), and diseasescaused by abnormal proliferation of fibrovascular or fibrous tissue,including all forms of proliferative vitreoretinopathy. An improvedability to monitor angiogenesis can assist in developing improvedmethods of intervention, diagnosis, and prognosis of such diseases.

[0021] Angiogenesis in Solid Tumor Formation and Metastasis.

[0022] Angiogenesis is prominent in solid tumor formation andmetastasis. Several experimental studies have concluded that primarytumor growth, tumor invasiveness, and metastasis all requireneovascularization. The process of tumor growth and metastasis iscomplex, involving interactions among transformed neoplastic cells,resident tissue cells (e.g., fibroblasts, macrophages, and endothelialcells), and recruited circulating cells (e.g., platelets, neutrophils,monocytes, and lymphocytes). A possible mechanism for the maintenance oftumor growth is an imbalance, or disregulation, of stimulatory andinhibitory growth factors in systems within the tumor. Disregulation ofmultiple systems allows the perpetuation of tumor growth and eventualmetastasis. Angiogenesis is one of many systems that is disregulated intumor growth. In the past it has been difficult to distinguish betweendisregulation of angiogenesis and disregulation of other systemsaffecting a developing tumor. As another complicating factor, Maniotis AJ et al. Am J Pathol September ;155(3):739-52 (1999) have noted thataggressive human melanomas mimic vasculogenesis by producing channels ofpatterned networks of interconnected loops of extracellular matrix, inwhich red blood cells, but not endothelial cells, are detected. Thesechannels may facilitate perfusion of tumors, independent of perfusionfrom angiogenesis.

[0023] A tumor cannot expand without a blood supply to provide nutrientsand remove cellular wastes. Tumors in which angiogenesis is importantinclude solid tumors, and benign tumors including acoustic neuroma,neurofibroma, trachoma and pyogenic granulomas. Inhibiting angiogenesiscould halt the growth of these tumors. Angiogenic factors have beenreported as being associated with several solid tumors, includingrhabdomyosarcoma, retinoblastoma, Ewing sarcoma, neuroblastoma, andosteosarcoma.

[0024] Angiogenesis has also been associated with some non-solid tumors,including blood-born tumors such as leukemias, various acute or chronicneoplastic diseases of the bone marrow marked by unrestrainedproliferation of white blood cells, usually accompanied by anemia,impaired blood clotting, and enlargement of the lymph nodes, liver, andspleen. It is believed that angiogenesis may play a role in theabnormalities in the bone marrow that give rise to leukemias.

[0025] Tumor Growth Beyond 1 to 2 mm Diameter is Dependent onAngiogenesis.

[0026] Angiogenesis in normal wound repair appears to be under strictcontrol, and is self-limited. By contrast, neovascularization isexaggerated and is not well-controlled during neoplastic transformation.It appears that tumors continually renew and alter their vascularsupply. Normal vascular mass is approximately 20% of total tissue mass,while tumor vascular mass may comprise as much as 50% of the totaltumor. Neovascularization is both a marker of pre-neoplastic lesions, aswell as a condition that perpetuates tumor growth.

[0027] Several studies have found a correlation between the magnitude oftumor-derived angiogenesis and metastasis in melanoma, prostate cancer,breast cancer, and non-small cell lung cancer. These studies support theconclusion that tumor-associated angiogenesis is disregulated, with animbalance that favors either the expression of local angiogenic factorsor the suppression of angiostatic factors. Also, the degree ofangiogenic response in a tumor is related to the prognosis; i.e., thehigher the degree of angiogenesis, the worse the prognosis.

[0028] Experimental Models of Angiogenesis.

[0029] A source of angiogenic stimulation can be either endogenous orexogenous to the vessel-sprouting tissue. Exogenous stimulation requirestwo types of tissue, the stimulating tissue and the responding orsprouting tissue. Endogenous stimulation requires only one tissue, sinceboth the stimulus and the response occur within the same tissue.

[0030] Several in vivo angiogenesis models have been developed. Thecorneal pocket assay involves the surgical implantation of polymerpellets containing angiogenic factors in the cornea of larger animalssuch as rabbits. Quantitation is difficult, and few such tests haveapparently been conducted. The chick chorioallantoic membrane assayinvolves the removal and transfer of a chick embryo from the shell to acup. The angiogenic material is suspended in a vehicle, typically asolution of methyl cellulose, and is then dried on a glass cover slipand placed on the chorioallantoic membrane. The appearance of newvessels is observed. The rabbit ear chamber assay requires the surgicalinsertion of a glass or plastic viewing device, and the measurement ofcapillary migration by microscopy. However, it is difficult to applyangiogenic materials in this assay. The rat dorsal air sac assayinvolves implants of stainless steel chambers containing angiogenicfactors and is difficult to quantitate. The alginate assay involves thesubcutaneous injection into mice of tumor cells encased in alginate.

[0031] The endothelial cell proliferation assay relies on measurementsof cell proliferation. It is typically performed in 96-well tissueculture plates.

[0032] The endothelial cell migration assay assesses migration ofendothelial cells toward a stimulus. Inhibition of angiogenesis is shownby blockage of migration in the presence of the inhibitor. See Dameronet al., Science, 265, 1582-84 (1994).

[0033] In the endothelial cell tube formation assay, human umbilicalvascular endothelial cells (HUVECs) are plated on gels of a matrix suchas Matrigel™. See Schnaper et al., J. Cell. Physiol, 156, 235-246(1993). Matrigel™ is described in U.S. Pat. No. 5,382,514. Baatout S,and Cheta N, Rom J Intern Med 1996 July-December ; 34(3-4):263-9describe Matrigel™ as a mixture of basement membrane proteins includinglaminin, type IV collagen, entactin/nitrogen and proteoheparan sulfate,and various growth factors. Matrigel™ induces endothelial cells todifferentiate, as evidenced both by morphologic changes and by areduction in proliferation. It therefore offers a convenient system tostudy biochemical and molecular events associated with angiogenesis.Further, Matrigel™ permits one to study the roles of the extracellularmatrix in angiogenesis. Sprouts from vessels in adjacent tissuepenetrate into the gel within days of connecting it to the externalvasculature.

[0034] Maldonado et al., Pathol Oncol Res; 4:225-9 (1998), developed anangiogenesis model that demonstrated that human metastatic prostatecancer cells appeared to induce HUVECs to translocate across aMatrigel-coated membrane.

[0035] The corneal micropocket assay is widely accepted as beinggenerally predictive of clinical usefulness. In this assay, anangiogenic agent is a factor that is seen to consistently act to promotethe ingrowth of one or more blood vessels within the cornea, preferablywithout evidence of the influx of leukocytes.

[0036] The rodent mesenteric-window assay is a model that exploits thevirtually avascular membranous rodent mesentery. After experimentaltreatment, angiogenesis is quantified in the mesentery histologically asthe number of vessels per unit length of mesentery. See Norrby et al.“Mast-cell-mediated angiogenesis: a novel experimental model”; VirchowsArch B Cell Pathol Incl Mol Pathol; 52:195-206 (1986).

[0037] In chemotactic chamber assays, millipore chambers containingtumors are implanted in an animal such as a hamster. Once such device isknown as a “Boyden chamber.” The Boyden chamber contains an upper welland a blind lower well, separated by a semipermeable membrane.Chemoattractants are placed in the lower well. See, e.g., U.S. Pat. No.4,912,057.

[0038] In the alginate-entrapped tumor cell assay, tumor cells entrappedin alginate are implanted in an animal. See Plunkett and Hailey,Laboratory Investigation, 62:510517 (1990).

[0039] In the microbead assay magnetic microbeads are incubated withcapillary endothelial cells, such that 10-15 microbeads are internalizedper cell. Cells containing the ingested beads are subjected to variousstimuli and allowed to proliferate, distributing the ingested beads intodaughter cells. Quantification and distribution of the average number ofbeads in individual cells allows one to monitor endothelial stimulationand inhibition. See Cao Y, et al., Lab Invest August ;78(8):1029-30(1998).

[0040] In a three-dimensional co-culture system, capillary-likestructures are induced in a structure containing sandwiched layers ofcollagen gels and fibrin gels. Each layer can be seeded with cells, suchas fibroblasts or cancer cells. It has been reported that in the absenceof fibroblasts, endothelial cells do not survive in this system. SeeJanvier et al. Anticancer Research 17:1551-1558 (1997).

[0041] There have also been exogenous models of angiogenesis using serumsupplements. Explants of muscular and adipose tissue, minced into smallfragments and embedded in a three-dimensional matrix of fibrin orcollagen, in the presence of serum, gave rise to an extensive outgrowthof branching and anastomosing capillary-like tubes. See Montesano et al.Cell Biology International Reports, 9: 869-875 (1985). This system wasnot autoregulatory, however, since regulatory substances were providedin the serum.

[0042] In each of these assays, tumors are modeled either by theactivity of single cells, or of a group of cells that induces theformation of blood vessels originating from tissue exogenous to theimplanted tumor, and then penetrating the tumor from without.

[0043] Endogenous Angiogenesis Models

[0044] By contrast to the exogenous angiogenesis assays described above,endogenous angiogenesis assays have been used to observe whetherparticular conditions promote the endogenous sprouting of new vesselsfrom tissue into a surrounding cell-free matrix in a serum-free medium.

[0045] One endogenous assay is the aortic ring assay. Preexisting bloodvessels can generate new vessels in the absence of exogenous angiogenicstimuli, because the vessel wall is autoregulatory through autocrine,paracrine, and juxtacrine mechanisms. (“Juxtacrine” signaling occurswhen the ligand and its receptors are both anchored in the cellmembrane.) The vessel wall produces growth factors, proteolytic enzymes,matrix components, cell adhesion molecules, and vasoactive factors.Thus, rat aortic or venous explants cultured in collagen gels underserum-free conditions will sprout new vessels induced by the combinedeffect of injury and exposure to collagen. See Nicosia R F, et al. IntRev Cytol, 185:1-43 (1999).

[0046] Another endogenous angiogenesis assay is the placental explantassay. The endometrium expresses interacting peptide and non-peptidegrowth factors during endometrial renewal, factors that includeepidermal growth factor, transforming growth factors (e.g. TGF-β),platelet-derived growth factor/thymidine phosphorylase, tumor necrosisfactors, and vascular endothelial growth factor (VEGF). See Smith S K,Hum Reprod Update 4:509-19 (1998).

[0047] In the angiogenesis assay described by Brown, et al. Lab Invest75:539-55 (1996), a fragment of human placental blood vessel embedded ina fibrin gel in microculture plates gave rise to a complex network ofmicrovessels during a period of 7 to 21 days in culture. This method isalso described in Australian patent AU-B 17500/95. This group hasrecently published a study of tumor inhibitors using this assay. SeeParish et al., Cancer Res; 59: 3433-41 (1999).

[0048] Prior Tumor Cell Angiogenesis Models Have Been Exogenous.

[0049] Unlike normal ovary, endometrium, and placenta, most tumor tissueis not specialized to function as an angiogenic organ. Neither doestumor tissue possess autoregulatory angiogenic capacity, as does theaorta. Thus, in all known prior models of tumor angiogenesis, the tumoris an angiogenic stimulus to which the surrounding tissue responds bysprouting new vessels toward and into the tumor. While tumor cellinvasion and angiogenesis share several similarities, there are alsoimportant differences. The initiation of both processes requiresattachment to a basement membrane, followed by disruption of themembrane and migration through the defect. After the invading cellcrosses the basement membrane barrier, cell proliferation produceseither a new vessel lumen or metastatic foci. It is likely that the twoprocesses are mutually stimulating, since vascularization allows tumorgrowth, and tumor growth requires vascularization. The two processesoperate in opposing directions, however. Tumor cell invasion occurs whencells move from a tumor into surrounding tissue, whereas tumor-inducedangiogenesis is the sprouting of new vessels from the surrounding tissuetoward the tumor.

[0050] Quantitating Angiogenesis.

[0051] Several methods have been used to quantitate angiogenesis orperfusion. See, e.g., Hoffman et al., Cancer Res September 1; 57(17):3847-51 (1997); and Cancer Res September 1;57(17):3847-51(1997). Okadaet al., Jpn J Cancer Res September ; 87(9): 952-7 (1996) described themeasurement of hemoglobin as a surrogate for direct angiogenesismeasurement.

[0052] Conrad et al., Lab Invest March ;70(3):426-34 (1994); Iwahana etal., Int. J Exp Pathol 77:109-14 (1996); Rohr et al., Nouv Rev FrHematol 34:287-94 (1992); and Nikiforidis et al., Eur J Radiol 29:168-79 (1999) disclose the use of computer image analysis to quantitateangiogenesis.

[0053] Matrices and Extracellular Matrices.

[0054] As used in the specification and claims, the term “matrix” refersto a porous, composite, solid or semi-solid substance, for example agel, having pores or spaces sufficiently large for cells to populate.Depending on context, the term “matrix” can also refer to matrix-formingmaterials, i.e., materials that will form a matrix under suitableconditions. Matrix-forming materials may, for example, require theaddition of a polymerizing agent to form a matrix, e.g., adding thrombinto a solution containing fibrinogen to form a fibrin matrix. Othermatrix materials include collagen (all types), combinations of collagenand fibrin, agarose (e.g., Sepharose™), and gelatin.

[0055] Extracellular matrices include, for example, collagen, fibrin,fibronectin, and hyaluronic acid. Artificial, biocompatibleextracellular matrices include, for example, dextran polymers, polyvinylchlorides, polyglycolic acids, polylactic acids, polylactic coglycolicacids, and silicone. Synthetic extracellular matrices are described inPutnam and Mooney, Nat Med 1996 Julu;2(7):824-6.

[0056] Matrices useful in the compositions and methods of this inventionmay be pre-formed. or they may be formed in situ, for example, bypolymerizing compounds and compositions such as fibrinogen to form afibrin matrix. Matrices that may be preformed include those made fromthe following components, or various mixtures of the followingcomponents: collagen, collagen analogs or collagen mimics (e.g.,collagen sponges and collagen fleece), chemically modified collagen,gelatin beads or sponges, gel-forming or composite substances comprisinga biocompatible matrix material that will allow cells to populate thematrix, and collagen complexed with other compounds to enhancecollagen's ability to polymerize, maintain its structure, or resistdegradation. See, e.g., U.S. Pat. Nos. 5,830,492; 5,824,331; 5,834,005;and 5,922,339.

[0057] In addition to fibrin gels, Matrigel, alginate, agarose, andbiological-molecule-impregnated polyester have been used as matrices toenhance angiogenesis. See Fournier and Doillon, Biomaterials 17:1659-65(1996). Zimrin A B et al., Biochem Biophys Res Commun 1995August15;213(2):630-8, noted that there were some differences betweenendothelial cells cultured in the presence of fibrin versus thosecultured in Matrigel.

[0058] U.S. Pat. No. 5,830,504 discloses an artificial bioactive matrixcomprising cooperative combinations of ligands within a matrix.

[0059] Kim B S et al., Biotechnol Bioeng January 1998 5; 57(1):46-54,describe the use of polyglycolic acid as an extracellular matrix.

[0060] Changes in extracellular matrix structure and composition canhave important regulatory effects on cell behavior. For example, Kanzawaet al., Ann Plast Surg March 1993; 30(3): 244-51, examined angiogenesisin a three-dimensional model in vitro, using HUVECs cultured in acollagen gel. An abundant, capillary-like network with a lumen structurewas seen histologically, forming at a collagen density less than 0.15%for either type I or type III collagen. At the same density, type IIIcollagen induced a capillary-like network with HUVECs at an earlierstage of culture than did type I collagen. Thus, both collagen densityand type can influence angiogenesis.

[0061] Endothelial growth medium is a serum-free medium that supportsthe growth and maintenance of vascular endothelial cells. See, e.g.,Gorfien et al. (1993) Exp. Cell Res. 206, 291; and Gorfien et al. (1992)Focus 14: 14. The high levels of serum supplementation that are oftenused in endothelial cell culture may create problems in experimentaldesign or in interpretation of results.

[0062] Gorman L et al., Nutrition April 1996; 12(4):266-70, furtherrefined the growth requirements of endothelial cells. These authorsreported that M199 medium that is deficient in amino acids butsupplemented with glutamine was superior to M199 complete medium (medium199 (Gibco BRL, Grand Island, N.Y.)).

[0063] U.S. Pat. Nos. 6,139,574 and 6,176,874 disclose solid free-form(e.g., polymeric) fabrication methods for manufacturing devices fortissue regeneration, in a matrix having a network of lumens said to befunctionally equivalent to the naturally occurring vasculature oftissue, which can be lined with endothelial cells and coupled to bloodvessels at the time of implantation.

[0064] Published international application WO 95/23968 discloses amethod for obtaining angiogenesis by culturing a blood vessel fragmentwith a physiological gel and nutrients. The physiological gel was saidto preferably be fibrin, collagen, Matrigel, or similar.

[0065] “Cell treatment could help doctors make old hearts young again,”internet article available athttp://www.cnn.com/2000/HEALTH/11/12/heart.repair/index.html (November2000) is an account in the popular press of treating damaged hearts byinjecting isolating skeletal myoblasts around the area of a scar on theheart tissue. Similar approaches using marrow stromal cells andcirculating immature endothelial cells were also mentioned.

[0066] No prior reports are known of angiogenesis assays for tumors orother tissue in which the intact three-dimensional structure of thetissue is maintained during the assay—as opposed to, for example,reports of an assay conducted on an isolated artery or vein.

[0067] No prior reports are known in which angiogenesis has beenpromoted in three-dimensional tissues ex vivo prior to transplantation.

[0068] We have discovered an in vitro tissue angiogenesis andvasculogenesis system that allows the outgrowth of microvessels from athree-dimensional tissue fragment implanted in a matrix. The matrix may,for example, be a fibrin- or collagen-based matrix fed by a growthmedium, for example, a mixture of tissue culture medium, serum, or alayer of serum-free medium with defined growth factors. This system,which may be used with human or other mammalian or animal tissues, maybe used in assaying tumor angiogenic potential, or in promotingangiogenesis in other tissues, e.g., promoting angiogenesis prior totransplantation of a tissue. The angiogenic potential of a tissue can bedetermined by measuring the growth of microvessels into the matrix. Thesystem is based on endogenous angiogenesis, vasculogenesis,neovascularization, or tissue perfusion, independent of tumorangiogenesis or other tissue angiogenesis. By contrast, tumorangiogenesis per se results from the formation of patterned networks ofinterconnected loops of extracellular matrix through which tumorperfusion may occur. The three-dimensional structure of the tumor orother tissue is maintained in the matrix, including its blood vessels,supportive stromal elements such as fibroblasts, and neural andendothelial cells. In another aspect, the method allows for theproliferation of a tissue specimen, thus increasing the mass of cellsavailable for subsequent transplant; and the method also provides forthe proliferation of blood vessels from the tissue mass, thus enhancingthe chance of successful engraftment. The mass of the tissue to betransplanted is preferably increased by at least about 25%, morepreferably by at least about 50%, most preferably by at least about100%.

[0069] Unless otherwise clearly indicated by context, the appearance ofnew vessels in the novel system, whether by angiogenesis orvasculogenesis, is considered as a measure of the angiogenic potentialof a tumor or other tissue. Classification as “angiogenesis,”“vasculogenesis,” or “neovascularization” may help promoteunderstanding, but should not be interpreted to limit the scope of thepresent invention. Moreover, for the purposes of the presentspecification and claims, unless otherwise clearly indicated by context,the term “angiogenesis” should be interpreted also to include theprocesses of vasculogenesis and neovascularization.

[0070] The novel system displays several unique and surprisingcharacteristics that are not found in any known prior tissueangiogenesis model. Intact tissue architecture is maintained, includingsupportive stromal elements (e.g., fibroblasts), neural tissues, andendothelial tissues. The inclusion of such elements is important, as thepresence of these tissues and of the supporting fibrin matrix betterprovide the framework required for angiogenesis and growth of tumors orother tissues. Vessel growth rate typically exceeds the rate of tissuegrowth, meaning that the growth rate of angiogenic vessels may bemeasured without interference from tissue growth. The ability toindependently and accurately measure the growth of angiogenic vessels isparticularly surprising, because no known prior model has provided thisimportant capability. The differential growth pattern of tissue cellsand angiogenic vessels in a fibrin gel matrix separates the angiogenicvessels and the tissue stroma into independently observable regions ofinterest (vessel and tissue compartments). The compartmental structureof the novel system allows the measurement of differential effects ofvarious anti-tumor or tissue stimulatory therapies on tissue andangiogenic vessel components.

[0071] The present invention may be used to observe angiogenesis in anytype of solid tumor, or to promote angiogenesis in any type of normal,vascularized tissue. If desired, results maybe expressed in asemi-quantitative or quantitative manner; quantification may beconducted, for example, by direct examination, computer-assisted imageanalysis, or measurements of surrogate indicators of the creation ofperfusion channels. Examples of such surrogate indicators includetritiated-thymidine uptake, gene up regulation, and¹²⁵I-bromodeoxyuridine uptake.

[0072] Methods of cell culture, gel formation, vessel quantitation, andmatrix preparation are well known in the art. Thus, most methods of cellculture or gel formation that will support growth of cells embeddedwithin a matrix may be used to practice the present invention, includingby way of example those described in the present application. Moreover,most matrices capable of supporting angiogenesis may be used to practicethe present invention, including by way of example those described inthe present application. Also, any method of vessel quantitation,including but not limited to those described in the presentspecification, may be used to practice the invention.

[0073] Test compounds, angiogenesis factors, or sera are preferablylayered over or incorporated into the feeding layer in an appropriateconcentration. The compounds or sera then diffuse into the fibrin matrixto produce effects on the tissue fragment and its sprouting angiogenicvessels.

[0074] Evaluation of Neovessel Initiation.

[0075] The initiation fraction may be computed by counting the number ofwells that develop an angiogenic response, and dividing by the totalnumber of wells plated.

[0076] Angiogenesis Initiation Rate.

[0077] The initiation rate equals the slope of the curve of a plot ofthe fraction of angiogenesis initiation in culture against time.

[0078] Evaluation of Neovessel Proliferation/Promotion.

[0079] For subjective scoring, the discs are divided into four quadrantsand rated on a 0-4 scale for the amount of angiogenic growth. Using a0-4 rating scale in each of four quadrants, a total score of 0-16 may bedetermined for each well. If desired, a more objective measurement maybe obtained, for example, by using optical microscopy and digital imageanalysis to measure the total surface area of angiogenic sprouting. Bymeasuring total surface area as a function of time, the rate of changemay be determined.

[0080] Viability Measurements.

[0081] Cellular viability may be evaluated using any of various methodsknown in the art. A convenient method is a colorimetric assay such asthe MTT assay (Promega, Madison, Wis.). This assay is based on thecellular conversion of a tetrazolium salt into a blue formazan product.The MTT assay can be performed at the end of a specified time period onboth the tissue fragment and on angiogenic sprouts. This assay can beused, for example, to compare drug/sera-treated and untreated wells.

[0082] Proliferation Measurements.

[0083] Any of various methods known to the art may be used to measureproliferation of cells. For example, uptake of nonspecific tracers suchas ³H-thymidine or ¹²⁵I-UDR, which incorporate only into activelydividing cells, may be used to compare uptakes in treated wells versusuntreated wells. Use of specific receptor-mediated tags can also be usedto assess tissue-versus-vessel uptake in treated and untreated wells.Statistically significant differences in uptake are attributed toeffects of the drug, serum, or other treatment.

[0084] Tumor and Other Tissue Sources.

[0085] Monolayer cell lines, solid tumor fragments, or other tissues maybe harvested from or grown in a suitable host animal. A suitable hostfor many experimental purposes is the nude mouse. Tumors, for example,are harvested upon reaching a size of 1-2 cm, which is sufficient toprovide an adequate number of tumor discs. For clinical purposes, freshsurgical specimens may be used to assess the angiogenic potential of aparticular tumor or other tissue. Exposing a cut surface within thetumor or other tissue, i.e., exposing cut blood vessels, is believed toenhance the tissue's angiogenic response by inducing hypoxia in thetransected vessel edges.

[0086] Assays.

[0087] The novel system may be used in various assays to test theeffects of different agents on angiogenesis. Examples of such agentsinclude growth factors, growth factor inhibitors, serum (includingautologous serum), chemotherapeutic agents, external beam radiation,in-situ radiation therapy (such as that delivered viaradiopharmaceutical targeting compounds, for example radiolabeledsomatostatin, monoclonal antibodies, and peptides), growth factors,growth factor inhibitors, steroid and peptide hormones or their analogs,and chemotherapeutic agents.

[0088] Monolayers of various tumor cells lines can be placed into oronto a solid/semi-solid feeder layer to test the effects on angiogenesisof mediators released from the cells.

[0089] In vitro Metabolic Manipulations.

[0090] The tissue-specific metabolism of different soluble substancesmay be evaluated by implanting cells, for example hepatocyte clusters orliver fragments, into the solid/semi-solid feeder layer. The effects ofsoluble factors in circulating blood may be evaluated by replacing theliquid feeder layer with serum, including autologous serum from the samepatient.

[0091] Non-Oncological Applications.

[0092] In addition to evaluating responses in tumors, this inventionallows the evaluation or the promotion of angiogenic responses in othertissues or organs undergoing physiologic or pathophysiologic changes.Such other applications include, for example, the evaluation ofembryologic tissues, the promotion of angiogenesis in wounds, in cardiacmuscle; or conversely the evaluation of the inhibition of angiogenesisin inflamed tissues of rheumatic disorders, or in skin conditions suchas psoriasis. Other applications include the induction of angiogenesisin a tissue transplant, including an autologous transplant; diseasessuch as parathyroid reimplantation in the forearm following totalparathyroidectomy, or reimplantation of pituitary, adrenal, pancreatic,other endocrine tissues, or other peptide- or amine-producing tissues.The inhibitors and stimulators of angiogenesis in any tissue may bestudied using an assay in accordance with the present invention. Tissuemay be allowed to grow in assay conditions until the host tissueproliferation increases significantly above the mass of tissueoriginally implanted in the system.

[0093] In Vivo Systemic Assays Using the Present Invention.

[0094] The present invention maybe used in conjunction with an in vivosystemic assay. Tumor growth is initiated in a suitable host such as thenude mouse or rat; the tumors are allowed to grow to 1-2 cm; and thetumors are then challenged systemically with the test compound orradiation treatment of interest. Following treatment, the animal issacrificed and a tumor is harvested. A tumor harvested prior to thesystemic test serves as a control. The tumors are both processed as perthe 3DTAM protocol (S. Gulec et al., “A new in vitro angiogenesis assaywith spatially intact human tumor architecture. The 3D tumorangiogenesis model (3DTAM), preprint 2001). Both sets of tumor fragmentsare evaluated for their angiogenic response. This approach allows one toassess the effects of in vivo therapy in the presence of biologicvariables that affect drug pharmacokinetics, such as liver metabolismand renal excretion, as well as humoral interactions at the plasma ortissue level.

[0095] Multi-Compartment Techniques.

[0096] Multiple compounds or radiation treatments can be evaluatedsimultaneously with multiple wells, separated from one another bydialysis membranes. Multi-compartment procedures can also be conductedwith compartments or wells comprising a non-toxic, water-soluble orwater-insoluble gel. Such gels include, for example, collagen, othercollagen-based materials as previous discussed, agarose, agar, alginate,silica, or protein-based gels such as gelatin. The wells are loaded withfibrin, or with a soft gel containing tissue samples. In thisembodiment, the compartments or wells may optionally be sealed, forexample with a layer of agarose, before the wells are filled. Adjacentwells may be used for sera, tumor, or tissue to provide comparativedata. A multi-compartment system separated by semipermeable membranes orgels may be used to evaluate the ability of a tumor, serum, or otherfactor to induce a directional angiogenic response. Optionally, one mayharvest all or a portion of the gel separating different wells. Theharvested portions may then be assayed for specific diffusiblesubstances responsible for inducing a directional angiogenic response.

[0097] Advantages of the Novel System

[0098] The invention allows a tumor or other tissue to induce anangiogenic response while maintaining an intact three-dimensionalarchitecture.

[0099] The present invention offers several advantages. It allows theevaluation of a tumor or other tissue's angiogenic response whilemaintaining an intact three-dimensional architecture. Tumor (or othertissue) compartments maybe evaluated simultaneously or separately. Thenovel system allows the evaluation of drugs that require activation invivo and drugs that are active ex vivo. One advantage of this inventionis that it may be used to provide a functional (as opposed tohistological) angiogenic index. A functional angiogenic index may helpto reveal tumors with a poor prognosis due to a high functionalangiogenic index, even though they may have a low histologicalangiogenic index. A disparity between functional and histologicalangiogenic indices may occur if circulating anti-angiogenic substances(such as angiostatin/endostatin) mask the angiogenic potential of atumor. Culturing tumors in a serum-free environment may better “unmask”angiogenic suppressors or stimulators, and thus better reveal their trueangiogenic potential. In lieu of a serum-free environment, a low serumenvironment (e.g., less than about 20% or less than about 10% serum) maybe used. This may demonstrate that removal or debulking of tumors thatsecrete a suppressor is not warranted and may be harmful.

[0100] Conversely, using this system in the presence of high serumlevels (greater than about 50% serum) may unmask angiogenesissuppressors that are present in some serum types, such as those fromnude mice implanted with Lewis lung carcinoma.

[0101] The invention may also be used to develop prognostic tests for apatient's resistance or susceptibility to the future development ofmalignancy or angiogenesis-related diseases.

[0102] An important aspect of the invention is its use in ex vivoangiogenesis to develop a blood supply in a tissue to be engrafted, thusdecreasing the time needed for adequate microcirculation to developafter implantation. This method also promotes the proliferation oftissue, which may increase the cell population available to engraftsubsequently. Such cell population increase may be desirable forimplantation of various tissues, for example endocrine tissue (e.g.,thyroid, adrenal glands, pancreas, pituitary, parathyroid), muscletissues (e.g., cardiac or skeletal muscle), kidney, liver, skin,prostate, retina, and other tissues.

[0103] The invention may also be used to evaluate the up or downregulation of a specific gene by a tumor or tissue, thus allowingtreatments to be based on gene expression.

EXAMPLES

[0104] As initial examples, we studied receptor mediated cytotoxiceffects of various radiolabeled somatostatin analogs.

[0105] This initial study observed significant in vitro cytotoxiceffects on human tumors and their angiogenic vessels by somatostatinanalogs labeled with ¹¹¹In, ¹²⁵I, or both.

[0106] We used the novel compartmental angiogenesis system to study thedifferential effects of somatostatin receptor subtype 2 (“sst-2”)mediated, in situ radiation therapy on tumors and their angiogenicvessels in a way that could not have been accomplished with priorangiogenesis models. The most dramatic results were obtained with IMR-32(human neuroblastoma) tumors, in which both the tumor and the vascularcompartments expressed sst-2. Tumor dissolution and angiogenic vesseldisruption were seen in all fragments that were treated with aradiolabeled somatostatin analog. Conversely, we observed no effect ofradiolabeled somatostatin analogs on the MDA (human breast carcinoma)tumor fragments. Watson, J. C. et al., Surgery August ; 122(2): 508-13(1997) demonstrated similar differences in the cytotoxicity ofsomatostatin analogs labeled with Auger emitters in tumor cell monolayercultures.

[0107] Somatostatin analogs containing an Auger electron-emitting labelprovided an excellent test of the invention. Auger electron treatmentrepresents true in situ radiation therapy, in which radiation isdelivered to a target following the specific high affinity binding of aradiolabeled ligand (e.g., a somatostatin analog) to its receptor (e.g.,a somatostatin receptor). Auger electrons emitted by radioisotopes suchas ¹¹¹In or ¹²⁵I have a very short range (on the order of 50 Å), and aretherefore only effective if the radioisotope can be deliveredintracellularly, preferably to the nucleus itself. The use of Augerelectron-emitting, targeted radiopharmaceuticals limits collateralradiation damage to normal cells by limiting cytotoxicity to those cellsthat bind and internalize the radioligand. Moreover, since thesomatostatin receptor sst-2 is uniquely overexpressed in angiogenicblood vessels, labeled somatostatin analogs will bind only to angiogenicblood vessels, but not to their normal counterparts.

[0108] We chose the somatostatin analogs ¹¹¹In-pentetreotide(Mallinckrodt Medical, St. Louis, Mo.), JIC2DL, and DTPA-JIC2DL. (Forthe latter two compounds, see D. Coy, W. Murphy, E. Woltering, J.Fuselier, and G. Drouant, “Hydrophilic Somatostatin Analogs,” U.S.patent application Ser. No. 09/196,259, filed Nov. 19, 1998.) Theanalogs were labeled with either ¹¹¹In or ¹²⁵I, or in some cases weredually labeled with both isotopes. JIC2DL has a sub-nanomolar bindingaffinity to the somatostatin receptor sst-2 (personal communication,David Coy, Tulane University, New Orleans, La.). JIC2DL can be iodinatedon its two-tyrosine residues, while DTPA-JIC2DL can be labeled with¹¹¹In, ¹²⁵I, or both.

[0109] We hypothesized that tumor xenograft explants expressing thesst-2 receptor would show cytotoxic changes when treated withradiolabeled somatostatin analogs, while those without sst-2 would not.We also hypothesized that treatment with radiolabeled somatostatinanalogs would inhibit angiogenic blood vessel growth, independent of thetumor's sst-2 status.

[0110] We cultured two human carcinoma cell lines obtained from theAmerican Tissue Culture Collection (ATCC). One cell line (IMR-32)expressed the sst-2 receptor and the other (MDA-MB-231 did not). Weimplanted these cell lines into nude mice to create human tumorxenografts. Subsequently we harvested the xenografts and embedded tumorfragments in fibrin gel matrixes. These tumor-containing gels weretreated with radiolabeled somatostatin analogs to determine whetherthese compounds would destroy tumor cells or angiogenic blood vessels.

[0111] We demonstrated that the IMR-32 human neuroblastoma cell lineexpressed sst-2 as expected from its neuroendocrine differentiation,while the MDA-MB-231 human breast adenocarcinoma cells did not expresssst-2. Angiogenic vessels also express sst-2, while other blood vesselsdo not. We tested the following two compartment pairs with these celllines: (1) sst-2 (+) tumor, sst-2 (+) neovessels; and (2) sst-2 (−)tumor, sst-2 (+) neovessels.

[0112] The human breast carcinoma cell line, MDA-MB-231, was maintainedin Lebowitz's L-15 medium (Life Technologies Inc, Grand Island, N.Y.),supplemented with 10% fetal bovine serum (FBS) (Life Technologies Inc,Grand Island, N.Y.). The human neuroblastoma cell line, IMR-32, wasmaintained in Minimum Essential Medium (Life Technologies, Inc, GrandIsland, N.Y.), supplemented with 15% FBS, non-essential amino acids(Life Technologies Inc, Grand Island, N.Y.), L-glutamine (Cellgro, Va.),and antibiotics. Cells were harvested at subconfluence and resuspendedin Hank's balanced salt solution (Life Technologies Inc, Grand Island,N.Y.).

[0113] While the initial examples described here report results obtainedwith fresh human surgical tumors or with tumors derived from tumor celllines, the same general technique will also work to promote angiogenesisex vivo in tissue explants intended for transplantation. Such tissuesmay, for example, be autologous, or they may be obtained during harvestfrom operative specimens, or brain dead donors—all in accordance withapplicable statutes, regulations, and Institutional Review Boardprocedures. The tissues will often proliferate in culture in parallelwith angiogenesis, further enhancing the usefulness of the tissue intransplantation. The ability to transplant intact tissues withpre-formed angiogenic vessels in this manner should provide substantialclinical benefits as compared to the infusion of individual cells, orthe transplant of tissue that has not been allowed to develop anangiogenic response.

[0114] Nude Mice and Creation of Human Tumor Xenografts.

[0115] All animal experiments reported in this specification wereapproved by the Louisiana State University Health Sciences Center animalcare committee. BALB/c Harlan Sprague Dawley nude mice (Indianapolis,Ind.) were injected with 1.5×10⁷ cells subcutaneously in both flankregions. The mice invariably grew solid tumors at the site of injectionover a period of 4-6 weeks. The tumors were allowed to reach a size of1.5-2 cm. (Continued growth to larger tumor sizes would often beaccompanied by central tumor necrosis.) Tumors were harvested using asterile technique under inhalation anesthesia with methoxyflurane. Themice were euthanized immediately after tumor harvest.

[0116] Preparation of Tumor Fragments.

[0117] Fresh tumors were processed immediately after harvesting. Tumorfragments 2 mm diameter and 1 mm thick were prepared, and then embeddedin a fibrin gel. The fibrin gels were prepared in 96 well-plates using aspecific tumor supporting medium as described below.

[0118] Preparation of the Tissue Supporting Medium.

[0119] A serum-free, basic growth medium comprising a balanced saltsolution, an antibiotic-antifungal solution, and an endothelial growthmedium was buffered to a pH of 7.4. Specifically, 9.5 g of medium 199(Gibco BRL, Grand Island, N.Y.) was dissolved in 980 mL deionized H₂O.10 mL of antibiotic-antimycotic solution (Gibco BRL, Grand Island, N.Y.)containing 10,000 U of penicillin base, 10,000 U of streptomycin baseand 25 μg of Amphotericin B was added. The pH was then adjusted byadding 2.2 g of Na HCO₃ (EM Science, Gibbston, N.J.), and was furthertitrated with 1N NaOH if needed to reach a pH of 7.4. This solution wasmixed with endothelial growth medium (EGM) (Gibco BRL, Grand Island,N.Y.) in a 3:1 ratio, and was sterilized by passing it through a0.22-micron filter.

[0120] Preparation of Fibrin Matrix Components for Tumor FragmentEmbedding.

[0121] A pro-coagulation solution was prepared by dissolving fibrinogen(0.12 g )(Sigma, St. Louis, Mo.) and 0.2 g of ε-amino caproic acid in 40mL endothelial growth medium. Human thrombin (2 μl) (Sigma, St. Louis,Mo.) was placed in the bottom of each well of a 96 well plate.Endothelial growth medium is a serum-free medium designed for the growthand maintenance of vascular endothelial cells. See Gorfien et al. (1993)Exp. Cell Res.206: 291; and Gorfien et al. (1992) Focus 14: 14.

[0122] Final Assembly of the Fibrin Matrix Tumor System, and Maintenanceof the Well-Plates.

[0123] One tumor disc was placed in the center of each thrombin-treatedwell. 0.2 mL procoagulation solution was carefully layered over thetumor fragments in each of the wells. Fibrin clot formation took placewithin 20-30 minutes at 37° C. The plates were kept at 37° C. in a 5%CO2/95% air humidified atmosphere.

[0124] Radiolabeled Somatostatin Analogs, Treatment and EvaluationProtocol.

[0125] The radiolabeled somatostatin analogs used in the experimentswith the IMR-32 tumor line were (1) ¹¹¹In pentetreotide (MallinckrodtMedical St. Louis, Mo.); (2) ¹¹¹In-DTPA JIC2DL; (3) ¹²⁵I-JIC2DL and (4)¹¹¹In- and ¹²⁵I-DTPA JIC2DL. Support medium containing theradiopharmaceutical was added over the fibrin clots in the well platesbearing the tumor fragments, at 100 μCi/well. Concentrations for the¹¹¹In-labeled analogs and the ¹²⁵I-labeled analogs were 7.2×10⁻⁹ M and3.9×10⁻⁸ M, respectively. Treatments were administered on the first dayof tumor implantation. Each treatment group contained 30 tumorfragments. IMR-32 tumor fragments were treated with all 4radiopharmaceuticals tested (i.e., n=(30/treatment group)×4 groups=120total). ¹¹¹In-DTPA JIC2DL was the only radiolabeled somatostatin analogused in the experiments with the MDA-MB-231 tumor line (n=30). Controlgroups were given the support medium only (n=30 for each of the twotumor types). Capillary sprouting was monitored visually for 14 days.

[0126] The percentage of wells in which new angiogenic vessel growthinitiated was observed. For subjective angiogenic scoring, the discswere divided into four quadrants and rated on a 0-4 scale for the amountof angiogenic growth. Using this 0-4 rating scale in each of fourquadrants, a total score of 0-16 was given for each tumor fragment. Themean ± standard deviation of the angiogenic score for each treatmentgroup was calculated. Means for control and treatment groups werecompared for statistical significance (P<0.05) using the two-tailedStudent t-test.

Results

[0127] Angiogenic Initiation.

[0128] The angiogenic initiation fraction for all cultures was similar,regardless of tissue type or treatment. Untreated IMR fragments (24/30;80%) and untreated MDA tumor fragments (25/30; 83%) demonstratedangiogenic growth. The initiation fraction in ¹¹¹In-DTPA-JIC2DL treatedIMR and MDA tumor fragments were 25/30 (83%) and 24/30 (80%)respectively. The differences in initiation fractions of treated anduntreated tumor fragments in both groups were not statisticallysignificant. The initiation fractions for the IMR tumor fragmentstreated with the other radiopharmaceuticals were not significantlydifferent from control, ranging from 21/30 (70%) to 25/30 (83%).

[0129] Angiogenic Response.

[0130] The endpoints used to evaluate the compartmental tumorangiogenesis system included the response of the tumor(regression/disintegration) and the angiogenic response. The angiogenicresponse endpoints comprised the total angiogenic score, the fullangiogenic response fraction, the angiogenic inhibition pattern,primary-secondary failure, and architectural disruption. Angiogenicscores were calculated for each group of tumor fragments on day 14 usinga visual rating system. Fragments that did not show angiogenicinitiation were excluded from this portion of the analysis. Meanangiogenic scores for the control groups of IMR and MDA tumor fragmentswere similar (11.9±3.3 and 12.4±3.9, respectively). In the treated IMRlines, groups were observed with severe architectural disruptionassociated with tumor necrosis. No comparable scoring, therefore, waspossible for this treatment. In the treated MDA group, the meanangiogenic score was 6.4±2.9. This score was significantly differentfrom the mean angiogenic score of the MDA control group (12.4±3.9)(p<0.0001). 20/24 (83%) of the tumor fragments in the MDA treatmentgroup showed architectural disruption and evidence of vesseldestruction.

[0131] Tumor Response.

[0132] All tumor fragments in the control groups for both tumor celllines remained intact on day 14. In the treated IMR groups, all tumorfragments showed degenerative changes ranging from vacuolization tonearly complete tumor lysis. No significant differences were seen amongthe anti-tumor effects of the 4 different radiopharmaceuticals. In thetreated MDA group, all tumor fragments remained intact, with no evidenceof cytotoxic changes.

[0133] The most dramatic results were seen with the IMR-32 tumors, inwhich both the tumor and the vascular compartments expressed sst-2.Tumor dissociation and angiogenic vessel degradation were seen in allfragments that received the experimental treatment. Conversely, noeffect was seen on the MDA tumor fragments. However, angiotoxicity wasseen in 92% of the experimental MDA fragments. Sparing of the MDA (sst-2negative) tumor compartment from the effects of the in situ radiationwas strong evidence of the highly selective nature of the Auger emittertreatment.

[0134] Definition

[0135] Any biological system will, in a literal sense, bethree-dimensional. However, as used in the specification and claims, atissue or tissue fragment is considered to be “three-dimensional” if ithas multiple layers of cells comprising blood vessels and other cells ofthe tissue, and if the architecture of the tissue or tissue fragment(including, for example, the blood vessels, supportive stromal elementssuch as fibroblasts, neural and endothelial cells) is substantiallyintact and has not been disrupted as compared to the comparable tissuein vivo. As examples, a tumor, tumor sample, other tissue, or othertissue sample is considered “three-dimensional” within the scope of thisdefinition if its structure has not been disrupted. It may be sliced orreduced in thickness, so long as multiple layers of cells are retained,and so long as the relative structure and relation of blood vessels andother cells to one another is maintained.

[0136] As examples, the following would not be considered“three-dimensional” within the scope of the above definition: anisolated vein; an isolated artery; isolated cells from a disrupted tumoror other tissue; or an agglomerations of cells grown in culture—even anagglomeration that has substantial thickness and is “three-dimensional”in the ordinary sense—if the agglomeration lacks the architecture of thecomparable tissue in vivo—such as an agglomeration of tumor cells grownin culture without any vascularization.

[0137] The complete disclosures of all references cited in thisspecification are hereby incorporated by reference. In the event of anotherwise irreconcilable conflict, however, the present specificationshall control. Also incorporated by reference are the completedisclosures of the following unpublished papers, none of which is priorart to the present invention: S. Gulec et al., “Antitumor andantiangiogenic effects of somatostatin receptor-targeted in situradiation with ¹¹¹In-DTPA-JIC2DL,” J. Surg. Res., vol.97, pp.131-137(2001); and S. Gulec et al., “Antiangiogenic treatment with somatostatinreceptor-mediated in situ radiation,” American Surgeon (in press, 2001);S. Gulec et al., “A new in vitro angiogenesis assay with spatiallyintact human tumor architecture. The 3D tumor angiogenesis model (3DTAM)(preprint 2001). Also incorporated by reference is the completedisclosure of the priority provisional application No. ______ (which isa conversion of nonprovisional application Ser. No. 09/580,894), filedMay 30, 2000.

We claim:
 1. A method for assaying angiogenesis ex vivo, said methodcomprising the steps of: (a) embedding a three-dimensional mammaliantissue sample in a matrix, wherein the tissue sample has at least onecut surface exposing blood vessels; (b) supplying to the embedded tissuesample a medium that supports the growth of the tissue sample; (c)incubating the embedded tissue sample in the medium for a timesufficient to allow angiogenic vessels, if any, to grow into the matrixsurrounding the tissue sample; and (d) observing or measuring theangiogenic vessels, if any, that grow into the matrix surrounding thetissue sample.
 2. A method as recited in claim 1, wherein the mediumcomprises a serum-free medium that supports the growth of the tissuesample; wherein the medium contains substantially no exogenousangiogenesis-enhancing factors and substantially no exogenousangiogenesis-suppressing factors.
 3. A method as recited in claim 1,wherein the medium comprises serum.
 4. A method as recited in claim 1,wherein the medium comprises an angiogenesis-enhancing factor.
 5. Amethod as recited in claim 4, wherein the angiogenesis-enhancing factoris selected from the group consisting of platelet-derived growth factor,vascular endothelial growth factor, epidermal growth factor, fibroblastgrowth factor, and transforming growth factor β.
 6. A method as recitedin claim 1, wherein the matrix comprises fibrin.
 7. A method as recitedin claim 1, wherein the matrix comprises collagen.
 8. A method asrecited in claim 1, wherein the matrix comprises gelatin.
 9. A method asrecited in claim 1, wherein the matrix comprises agarose, agar,alginate, or silica gel.
 10. A method as recited in claim 1, wherein thematrix comprises Matrigel.
 11. A method as recited in claim 1, whereinthe tissue sample is a tumor fragment.
 12. A method as recited in claim1, wherein the tissue sample is not a tumor fragment, and wherein thetissue sample is not an isolated segment of an artery or vein.
 13. Amethod as recited in claim 1, additionally comprising the step ofsupplying an additional factor to the embedded tissue sample, andmeasuring the difference in angiogenesis for the tissue sample ascompared to the angiogenesis of an otherwise identical and otherwiseidentically-treated control tissue sample that is not supplied with thefactor; whereby the difference in observed angiogenesis is a measure ofthe angiogenic enhancement or angiogenic suppression characteristics ofthe supplied factor.
 14. A method for growing a tissue ex vivo, saidmethod comprising the steps of: (a) embedding a three-dimensionalmammalian tissue sample in a matrix, wherein the tissue sample has atleast one cut surface exposing blood vessels; (b) supplying to theembedded tissue sample a medium that supports the growth of the tissuesample; and (c) incubating the embedded tissue sample in the medium fora time sufficient to allow angiogenic vessels to grow into the matrixsurrounding the tissue sample; and to allow the number of cells in thetissue to proliferate, so that the tissue's suitability for transplantis improved.
 15. A method as recited in claim 14, wherein the mediumcomprises serum.
 16. A method as recited in claim 14, wherein the mediumcomprises an angiogenesis-enhancing factor.
 17. A method as recited inclaim 16, wherein the angiogenesis-enhancing factor is selected from thegroup consisting of platelet-derived growth factor, vascular endothelialgrowth factor, epidermal growth factor, fibroblast growth factor, andtransforming growth factor β.
 18. A method as recited in claim 14,wherein the matrix comprises fibrin.
 19. A method as recited in claim14, wherein the matrix comprises collagen.
 20. A method as recited inclaim 14, wherein the matrix comprises gelatin.
 21. A method as recitedin claim 14, wherein the matrix comprises agarose, agar, alginate, orsilica gel.
 22. A method as recited in claim 14, wherein the matrixcomprises Matrigel.
 23. A method as recited in claim 14, wherein thetissue sample is selected from the group consisting of skin tissue,parathyroid tissue, thyroid tissue, pituitary tissue, adrenal tissue,pancreas tissue, cardiac muscle tissue, skeletal muscle tissue, retinatissue, kidney tissue, liver tissue, and prostate tissue.
 24. A methodas recited in claim 14, additionally comprising the subsequent step oftransplanting the incubated embedded tissue sample with angiogenicvessels into a host in need of such a transplant.
 25. A method asrecited in claim 14, wherein said incubating step is conducted for atime sufficient for the mass of the tissue to increase by at least about25%.
 26. A method as recited in claim 25, additionally comprising thesubsequent step of transplanting the incubated embedded tissue samplewith angiogenic vessels into a host in need of such a transplant.
 27. Atissue with angiogenic vessels produced by the method of claim
 14. 28. Atissue with angiogenic vessels produced by the method of claim
 15. 29. Atissue with angiogenic vessels produced by the method of claim
 16. 30. Atissue with angiogenic vessels produced by the method of claim
 17. 31. Atissue with angiogenic vessels produced by the method of claim
 18. 32. Atissue with angiogenic vessels produced by the method of claim
 19. 33. Atissue with angiogenic vessels produced by the method of claim
 20. 34. Atissue with angiogenic vessels produced by the method of claim
 21. 35. Atissue with angiogenic vessels produced by the method of claim
 22. 36. Atissue with angiogenic vessels produced by the method of claim
 23. 37. Atissue with angiogenic vessels produced by the method of claim 25.